EDTA enhanced plant growth, antioxidant defense system, and phytoextraction of copper by Brassica napus L. Ume Habiba, Shafaqat Ali, Mujahid Farid, Muhammad Bilal Shakoor, Muhammad Rizwan, Muhammad Ibrahim, Ghulam Hasan Abbasi, et al. Environmental Science and Pollution Research ISSN 0944-1344 Volume 22 Number 2 Environ Sci Pollut Res (2015) 22:1534-1544 DOI 10.1007/s11356-014-3431-5
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Author's personal copy Environ Sci Pollut Res (2015) 22:1534–1544 DOI 10.1007/s11356-014-3431-5
EDTA enhanced plant growth, antioxidant defense system, and phytoextraction of copper by Brassica napus L. Ume Habiba & Shafaqat Ali & Mujahid Farid & Muhammad Bilal Shakoor & Muhammad Rizwan & Muhammad Ibrahim & Ghulam Hasan Abbasi & Tahir Hayat & Basharat Ali
Received: 17 April 2014 / Accepted: 7 August 2014 / Published online: 28 August 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Copper (Cu) is an essential micronutrient for normal plant growth and development, but in excess, it is also toxic to plants. The present study investigated the influence of ethylenediaminetetraacetic acid (EDTA) in enhancing Cu uptake and tolerance as well as the morphological and physiological responses of Brassica napus L. seedlings under Cu stress. Four-week-old seedlings were transferred to hydroponics containing Hoagland’s nutrient solution. After 2 weeks of transplanting, three levels (0, 50, and 100 μM) of Cu were applied with or without application of 2.5 mM EDTA and plants were further grown for 8 weeks in culture media. Results showed that Cu alone significantly decreased plant growth, biomass, photosynthetic pigments, and gas exchange characteristics. Cu stress also reduced the activities of antioxidants, such as superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), and catalase (CAT) along with protein contents. Cu toxicity increased the concentration of reactive oxygen species (ROS) as indicated by the
increased production of malondialdehyde (MDA) and hydrogen peroxide (H2O2) in both leaves and roots. The application of EDTA significantly alleviated Cu-induced toxic effects in B. napus, showing remarkable improvement in all these parameters. EDTA amendment increased the activity of antioxidant enzymes by decreasing the concentrations of MDA and H2O2 both in leaves and roots of B. napus. Although, EDTA amendment with Cu significantly increased Cu uptake in roots, stems, and leaves in decreasing order of concentration but increased the growth, photosynthetic parameters, and antioxidant enzymes. These results showed that the application of EDTA can be a useful strategy for phytoextraction of Cu by B. napus from contaminated soils. Keywords Antioxidants . Biomass . Copper . EDTA . Phytoextraction . Tolerance
Introduction Responsible editor: Elena Maestri U. Habiba : S. Ali (*) : M. Farid : M. B. Shakoor : M. Rizwan : M. Ibrahim Department of Environmental Sciences, Government College University, Allama Iqbal Road, Faisalabad 38000, Pakistan e-mail: [email protected]
G. H. Abbasi Department of Soil Science, University College of Agriculture and Environmental Sciences, TheIslamia University of Bahawalpur, Bahawalpur, Pakistan T. Hayat Department of Environmental Sciences, COMSATS Institute of Information Technology, CIIT Abbottabad Campus, University Road, Tobe Camp, Abbottabad 22060, Pakistan B. Ali Institute of Crop Science and Zhejiang Key Laboratory of Crop Germplasm, Zhejiang University, Hangzhou 310058, China
High levels of copper (Cu) in agricultural soils result not only from natural sources but also from industrial activities, use of pesticides and fungicides, as well as from the application of Cu-rich pig and poultry slurries (Yruela 2005; Legros et al. 2010; Nagajyoti et al. 2010; Mackie et al. 2012; Farid et al. 2013). Cu is an essential micronutrient and is required for normal plant growth and development which plays an important role in photosynthetic electron transport and also acts as cofactor of several enzymes such as superoxide dismutase and ascorbate oxidase (Marschner 1995; Yruela 2005). However, Cu is highly phytotoxic at slightly higher concentrations than those required for optimal plant growth and inhibits various physiological functions in plants (Monnet et al. 2006; Gajewska and SkŁodowska 2010; Azooz et al. 2012). Furthermore, Cu toxicity induces oxidative stress by producing reactive oxygen species (ROS) such as superoxide radicals
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(O2−.), hydrogen peroxide (H2O2), and malondialdehyde (MDA) which further increase the production of lipids and proteins oxidation (Zhang et al. 2007; Zhao et al. 2010). Therefore, to avoid Cu toxicity in plants, Cu-contaminated soils should be remediated to reduce the Cu concentration in these soils to a desired/acceptable level. The use of green plants for remediation of heavy metalcontaminated soils is proposed as a cost-effective and extensively applicable technique (Raziuddin et al. 2011). Phytoextraction uses high biomass producing plants to remove toxicants from soil by accumulating them in harvestable tissues such as stems and leaves (Pilon-Smits 2005; Shakoor et al. 2013). However, efficiency of plants to uptake toxic metals varies with soil type, plant species, and environmental conditions (Metwally et al. 2005; Jadia and Fulekar 2008; Yang et al. 2010; Ehsan et al. 2014). Various plant species, known as hyperaccumulators, have been used for the phytoremediation of metal-contaminated soils. Recently, researchers have evaluated that many species of Brassica hyperaccumulate metals such as Cu, Cd, and Zn (Singh and Agrawal 2010; Szczygłowskan et al. 2011). Brassicas have faster growth rate and higher biomass above ground (Meng et al. 2009; Vangronveld et al. 2009; Vamerali et al. 2010; Kanwal et al. 2014). Brassica napus is one of the most productive oilseed crops among Brassica species and is cultivated worldwide (Szczygłowskan et al. 2011; Bareen 2012; Park et al. 2012; Ehsan et al. 2013). It has been reported that B. napus tolerates Cu stress better than Brassica juncea through specific physiological and biochemical activities (Feigl et al. 2013), and various genotypes of B. napus are easily available (Wenzel et al. 2003). However, B. napus response to Cu stress depends upon stress exposed and duration of the stress (Peško and Kráľová 2013). Thus, metalaccumulating ability of B. napus coupled with the potential to produce large shoot biomass and source of oil makes this plant an ideal candidate for phytoextraction, especially Cu, with the aim to produce biofuel due to increasing oil prices in the near future. However, studies are scarce concerning the Cu uptake and physiological response of B. napus under Cu stress (Park et al. 2012; Peško and Kráľová 2013). Success of phytoremediation can be limited by many factors including complexation with organic matter and soil characteristics such as clay content, pH, and cation exchange capacity (Salt et al. 1995; Quartacci et al. 2000). Chemically enhanced phytoextraction of metals has been the recent attention (Evangelou et al. 2007; Farid et al. 2013). Different chelating agents have been used to enhance metal solubility in the soil. Ethylenediaminetetraacetic acid (EDTA) is one of the most effective chelating agents for artificially increasing solubility, complexation, and uptake of Cu and several other toxic metals (Bareen 2012; Chigbo and Batty 2013; Kambhampati 2013; Mani et al. 2014). Thus, the addition of EDTA into the soil induces uptake and translocation of heavy
metals from the roots to shoots of plants (Meers et al. 2008) and is especially important for increased uptake and translocation of toxic metals. Cu, in general, concentration of plants is regulated internally rather than externally. However, mechanisms behind EDTA-assisted Cu phytoextraction have not been comprehensively investigated so far, especially in B. napus despite of the fact that this plant is metal hyperaccumulator. Therefore, in this study, we attempted to determine the promoting role of EDTA on different morphophysiological and biochemical parameters of B. napus and also to check its phytoextraction ability and tolerance under Cu stress.
Material and methods Growth conditions and treatments Healthy seeds of B. napus genotype (Faisal Canola) were thoroughly washed with distilled water and sown in trays containing two inches of sterilized sand, incubated at 20– 22 °C in growth chamber. After 4 weeks, the morphologically uniform seedlings were wrapped with foam at root shoot junction and then transferred in thermopore sheets, having holes in them, floating on the water in iron tubs of 40-L capacity with complete randomized design (CRD). Hoagland’s nutrient solution was supplied to plants with the composition as follows (μmol L−1): KNO3, 3,000; Ca(NO3)2, 2,000; KH2PO4, 100; MgSO4, 1,000; H3BO3, 50; MnCl2·4H2O, 0.05; ZnSO4·7H2O, 0.8; CuSO4·5H2O, 0.3; H2MO4·H2O, 0.10; and FeNa-CA, 12.5. Iron tubs were lined with polythene sheet. Aeration was done continuously by using air pumps. The solution was renewed after every 7 days. After acclimatization period of 2 weeks, plants were treated with CuSO4·5H2O and EDTA as Cu (50 μM), Cu (100 μM), EDTA (2.5 mM), Cu (50 μM)+EDTA (2.5 mM), and Cu (100 μM)+EDTA (2.5 mM) with three replications for each, whereas in control, no CuSO4·5H2O and EDTA was added. We used 2.5 mM EDTA and 50 and 100 μM Cu in the nutrient solution as these concentrations have already been used in the literature such as Azhar et al. (2006) and Zhao et al. (2010), respectively. EDTA was applied at juvenile stage as already used previously by Chigbo and Batty (2013). The pH was maintained at 6.0±0.1 throughout the experiment when required with 1 M H2SO4 or NaOH. Determination of plant growth parameters After 8 weeks of treatments, plant growth parameters were measured regarding plant height, leaf area, roots and shoot length, and number of leaves per plant along with fresh weight of roots, stems, and leaves. All plant parts were dried in an oven at 70 °C until constant weight and then weighed.
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Pigment analysis and SPAD value Chlorophyll (a and b) and carotenoid contents were extracted from a known fresh weight, topmost fully expanded leaves, of leaves in the dark with 85 % (v/v) aqueous acetone solution by continuous shaking until color had completely disappeared from the leaves. After centrifugation (4,000 rpm for 10 min at 4 °C), the supernatant was taken and 85 % aqueous acetone solution was added to dilute the solution to appropriate concentration for spectrophotometric measurements. Light absorbance at 663, 644, and 452.5 nm was measured by spectrophotometer (Metzner et al. 1965). The concentrations of chlorophyll and carotenoids were calculated by using the adjusted extinction coefficients (Lichtenthaler 1987). The SPAD (soilplant analyses development) is a faster way to collect chlorophyll readings. After 8 weeks of treatments, SPAD-502 (Zheijang Top Instruments Co., Ltd., China) meter was used to measure these readings. Gas exchange characteristics After 8 weeks of Cu stress, infrared gas analyzer (IRGA; CI340, Analytical Development Company, Hoddesdon, England) was used for the measurements of photosynthetic rate (A), transpiration rate (E), stomatal conductance (gs), and water use efficiency (A/E). Antioxidant enzymes and protein content measurement After 8 weeks of Cu stress, fully stretched leaves of plants and roots samples were collected for enzymatic analysis. Leaves and roots were chomped with mortar and pestle in liquid nitrogen. This assay was standardized in 0.05 M phosphate buffer (maintaining pH at 7.8) and filtered through four layers of muslin cloth and then centrifuged at 12,000×g at 4 °C for 10 min. Finally, this enzyme extract was stored and used for estimating the activities of different enzymes. Antioxidant enzymes including superoxide dismutases (SOD), guaiacol peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), and soluble protein in roots and leaves were measured by spectrophotometer. The content of soluble protein was analyzed according to Bradford (1976) using coomassie brilliant blue G-250 as dye and albumin as a standard. For the assessment of SOD activity, homogenized the samples in a medium having buffer of 50 mM potassium phosphate with pH 7.0 and 0.1 mM of EDTA and 1 mM dithiothreitol (DTT) as described by Dixit et al. (2001). The SOD activity was evaluated by assessing its power to subdue the photochemical decrease of nitrobluetetrazolium (NBT) by adopting the methodology of Giannopolitis and Ries (1977). One unit of SOD activity was determined as the quantity of
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enzyme that induced 50 % prohibition of photochemical reduction of the NBT. For the assessment of POD, samples were homogenized in a potassium phosphate buffer (50 mM), 0.1 mM EDTA, and 1 mM DTT. For POD assessment, the mixture (3 ml) of 50 mM phosphate buffer, 20 mM guaiacol, 40 mM H2O2, and 0.1 ml of enzyme extract was used. The chemical reaction was started by adding up the extract of enzyme (modified from Chance and Maehly 1955). The change in wavelength of the solution at 470 nm was measured by spectrophotometer (Halo DB-20/DB-20S, Dynamica, UK). CAT (EC 126.96.36.199) activity was determined according to Aebi (1984). The assay mixture (3.0 ml) was composed of 100 μl enzyme extract, 100 μl H2O2 (300 mM), and 2.8 ml 50 mM phosphate buffer with 2 mM CA (pH 7.0). The CAT activity was assayed by monitoring the decrease in the absorbance at 240 nm as a consequence of H2O2 disappearance (ε=39.4 mM−1 cm−1). APX (EC 188.8.131.52) activity was assayed according to the method of Nakano and Asada (1981). The reaction mixture consisted of 100 μl enzyme extract, 100 μl ascorbate (7.5 mM), 100 μl H2O2 (300 mM), and 2.7 ml 25 mM potassium phosphate buffer with 2 mM CA (pH 7.0). The oxidation activity of ascorbate was determined by the change in wavelength at 290 nm (ε=2.8 mM−1 cm−1). Determination of electrolyte leakage, malondialdehyde, and hydrogen peroxide Electrolyte leakage was measured according to Dionisio-sese and Tobita (1998). After treatment of 8 weeks, the uppermost fully extended leaves were cut into small parts of 5 mm in length and positioned in test tubes in which there was 8 ml of deionized and distilled water. The tubes were processed in incubator in water bath at 32 °C for 2 h, then electrical conductivity of initial medium (EC1) was assessed by using pH/conductivity Model 720, INCO-LAB Company, Kuwait. These samples were placed in autoclave at 121 °C for 20 min, then cooled to 25 °C and again, electrical conductivity (EC2) was measured and computed with the following formula. EL ¼ ðEC1=EC2Þ 100: The level of lipid peroxidation was measured in terms of malondialdehyde (MDA; a product of lipid peroxidation) content by the thiobarbituric acid (TBA) reaction using the method of Heath and Packer (1968), with minor modifications as described by Dhindsa et al. (1981) and Zhang and Kirkham (1994). A 0.25-g leaf sample was homogenized in 5 ml of 0.1 % TCA, then centrifuged at 10,000×g for 5 min. Four milliliters of 20 % TCA containing 0.5 % TBA was added in 1 ml aliquot of the supernatant. The mixture was heated at
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95 °C for 30 min and then quickly cooled in an ice bath. After centrifugation at 10,000×g for 10 min, the absorbance of the supernatant at 532 nm was measured and the value for the nonspecific absorption at 600 nm was subtracted. The MDA content was calculated by using an extinction coefficient of 155 mM−1 cm−1. Hydrogen peroxide (H2O2) was extracted by homogenizing 50-mg leaf or root tissues with 3 ml of phosphate buffer (50 mM, pH 6.5), then centrifuged at 6,000×g for 25 min. To measure H2O2 content, 3 ml of extracted solution was mixed with 1 ml of 0.1 % titanium sulfate in 20 % (v/v) H 2 SO 4 and the mixture was then centrifuged at 6,000×g for 15 min. The intensity of the yellow color of the supernatant was measured at 410 nm. Hydrogen peroxide content was computed by using the extinction coefficient of 0.28 μmol−1 cm−1.
Measurement of copper content Known weight of dry plant sample (0.5 g) was taken in a 100ml flask and then added 15 ml of concentrated HNO3. After mixing, the sample flasks were placed on a hot plate having temperature increased up to 275 °C and dens yellow fumes appeared from the flask. When quantity of dens yellow fumes become low, then hydrogen peroxide was added until dens yellow fumes disappeared. When samples became colorless, then flask were removed from the hot plate and the volume was made up to 25 ml by using distilled water. Cu contents in root, stem, and leaf were determined by using flame atomic absorption spectrometry (AAS) made of (novAA 400 Analytik Jena, Germany). The concentration and accumulation of Cu in plant root, stem, and leaf was measured by the following formula:
Cu concentration mg kg−1 DW ¼ reading of AAS dilution factor=dry wt: of plant part
The accumulation of Cu in plant shoot and root was estimated by the following formula: Cu accumulation mg plant−1 ¼ conc: of Cu dry wt: of plant organ
Statistical analysis Data presented are means of three replicates. Analysis of variance (ANOVA) was done by using a statistical package, SPSS version 16.0 (SPSS, Chicago, IL) followed by Tukey’s post hoc test between the means of treatments to determine the significant difference.
Chlorophyll contents and SPAD value A significant decrease in chlorophyll a (Chl-a) and chlorophyll b (Chl-b), total chlorophyll, and carotenoid concentrations was observed in the leaves of B. napus with increasing Cu concentration in solution as compared to control (Fig. 1). Application of 2.5 mM EDTA significantly increased shoot pigment concentrations under the Cu stress as compared to Cu-treated alone conditions. The highest chlorophyll and carotenoid concentrations were observed in leaves with EDTA treatment alone, while lowest values were observed in 100 μM Cu treatment. Similar trend was also observed for SPAD value in the leaves of B. napus that higher Cu concentration caused more reduction in SPAD value, while maximum SPAD value was recorded in EDTA alone. Gas exchange attributes
Results Plant growth and biomass Increasing Cu concentrations reduced plant growth parameters of B. napus including root length, plant height, number of leaves par plant, leaf area, fresh and dry weights of leaves, and roots (Table 1). Furthermore, this reduction was more obvious at higher Cu concentration (100 μM) confirming as dose-dependent interaction of plants and stress. The application of EDTA improved these growth parameters by reducing the inhibitory effects of Cu at both levels of Cu stress.
A significant decrease was observed in net photosynthetic rate, transpiration rate, stomatal conductance, and water use efficiency of B. napus at both level of Cu stress as compared to controls, and this reduction was found as dose-dependent (Fig. 2). Moreover, results showed that EDTA treatment significantly increased these gas exchange attributes under both levels of Cu stress. Activities of antioxidant enzymes and protein content The activities of SOD, POD, CAT, APX, and soluble protein content in the leaves and roots of B. napus exposed to Cu
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31.16±2.24a 31.37±3.31a 20.85±1.27c 25.36±1.63b 12.14±0.09e 15.48±0.39d
4.61±0.32a 4.96±0.35a 2.52±0.21c 3.44±0.08b 1.57±0.12e 1.95±0.15d
66.30±3.44a 66.60±4.31a 38.80±1.16c 47.73±1.35b 22.51±1.26e 29.14±1.05d
5.47±0.29a 5.79±0.17a 3.53±0.15c 4.41±0.11b 2.23±0.16e 2.82±0.08d
stress alone or along with EDTA are given in Fig. 3. Apparently, activities of these enzymes were higher in leaves than in roots, except for SOD activity which was higher in roots than the leaves. The activities of SOD, POD, CAT, and APX significantly increased under both levels of Cu stress as compared to control and EDTA alone. Higher concentration of Cu (100 μM) significantly decreased the activity of these enzymes in both leaves and roots as compared to lower Cu concentration (50 μM) irrespective of EDTA addition. EDTA application, under both levels of Cu stress, significantly increased the activity of these enzymes as compared to same Cu treatment without EDTA. Protein contents were reduced significantly in leaves and roots of B. napus as Cu concentration increased in the solution (Fig. 3i, j). The application of EDTA significantly increased protein contents in both leaves and roots than that of the respective Cu treatment alone. Hydrogen peroxide, malondialdehyde, and electrolyte leakage
3.49±0.29a 3.56±0.26a 2.25±0.10c 2.72±0.11b 1.30±0.050e 1.76±0.85d
A significant increase was observed in H2O2, MDA, and electrolyte leakage levels in leaves and roots of B. napus under both levels of Cu concentrations as compared with control (Fig. 4). The exogenous application of EDTA under Cu stress caused a significant reduction in these parameters than that of Cu treatments alone.
28.37±1.31a 28.79±1.02a 19.73±1.14c 23.04±1.04b 11.60±1.30e 14.77±1.07d
Copper content in plant
Variants possessing the different letter are statistically significant at P>0.05
Control 12.06±0.34a 49.38±0.84a 13.74±0.70a 16.33±0.15a EDTA 12.36±0.31a 50.3±1.4a 13.63±0.86a 16.33±0.16a Cu50 6.48±0.16c 28.65±0.88c 6.51±0.53c 13.16±0.18c Cu50+EDTA 8.54±0.27b 38.63±0.83b 9.09±0.17b 14.8±0.11b Cu100 3.08±0.19e 12.82±2.09e 3.12±.58e 9.43±0.14e Cu 100+DTA 4.79±0.15d 19.87±0.5d 4.95±0.14d 11.63±0.09d
No. of leaves plant−1 Root fresh weight Root dry weight Stem fresh weight Stem dry weight Leaf fresh weight Leaf dry weight
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Table 1 Effect of Cu and EDTA on root length (cm), plant height (cm), leaf area (cm−1), and number of leaves per plant and fresh and dry biomass of roots, stems, and leaves (g plant−1) of Brassica napus L
Cu concentration in leaves, stems, and roots increased significantly with an increase in the level of applied Cu (Fig. 5a). The Cu concentration in roots was higher followed by stems and leaves, respectively. EDTA treatment further increased significantly Cu concentrations in all plant parts than that of control or respective Cu treatments alone. The accumulation of Cu in shoots was higher than roots, and shoot to root ratios also increased significantly with EDTA application under Cu stress as compared to respective Cu treatments alone (Fig. 5b).
Discussion Effect of copper on plant physiology and plant composition Our results showed that B. napus growth and biomass decreased under Cu stress (Table 1). Toxic effects of Cu on plant growth and biomass have already been observed in many plants (Monnet et al. 2006; Gajewska and SkŁodowska 2010; Zhao et al. 2010; Azooz et al. 2012). The reduction in plant growth and biomass under Cu stress might be due to larger Cu concentration and accumulation in different plant parts (Fig. 5) that have been shown to induce phytotoxic effect
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0 1.6 1.4
Chlorophyll b (mg g-1 FW)
Total Carotenoids (mg g-1 FW)
Chlorophyll a (mg g-1 FW)
Total Chlorophyll (mg g-1 FW)
Fig. 1 Effect of Cu and EDTA on chlorophyll a and b (a, b), total chlorophyll and total carotenoids (c, d), and SPAD value (e) in leaves of B. napus seedlings grown in solution culture with increasing Cu concentrations (0, 50, and 100 μM) treated without and with 2.5 mM EDTA. Different letters indicate that values are significant different at P